Morphometric changes on dung beetle Dichotomius problematicus (Coleoptera: Scarabaeidae: Scarabaeinae) related to conversion of forest into grassland: A case of study in the Ecuadorian Amazonia

Abstract The conversion of forest into grassland can induce differentiation in the functional morphology of resilient species. To assess this effect, we have chosen a dung beetle Dichotomius problematicus, as a model species. We established 20 sampling points distributed along a transect for a forest and grassland located in the Podocarpus National Park in Ecuador. Four pit‐fall traps were baited with pig feces per sample point and were left open for 48 h. We sexed and measured 13 morphological traits of 269 individuals. Nonmetric multidimensional scaling was carried out to evaluate the influence of habitat and sexual dimorphism on the traits. We applied a principal component analysis to evaluate the morphological features that best explain the differences between land use and sexual dimorphism. We used generalized linear models to evaluate the explanatory variables: habitat and sexual dimorphism with respect to morphological traits. Five traits contributed over 70% body thickness, Pronotum width, Pronotum length, Head width and Elytra length, following the results of a principal component analysis. Both habitat and sex influence traits. In the forest, the individuals are larger than grassland likely due to available resources, but in grassland, the structures in charge of the burial process head, protibia are larger, displaying a strong pronotum and possible a greater reproductive capacity given by spherecity. These patterns of changes in the size of beetles and their structures could reflect the conservation state of an ecosystem.


| INTRODUC TI ON
Increased land conversion of ecosystems from natural to anthropogenic states as a significant driver of decreasing taxonomic biodiversity has been extensively studied, especially at the community level (Carrión-Paladines et al., 2021;Edwards et al., 2014;Raine et al., 2018;Seibold et al., 2019). However, little is known about shifts in functional diversity with land conversion. There is evidence that the type of land use influences phenotypic plasticity in tropical dung beetles (Raine et al., 2018;Soto et al., 2019). These shifts could be observed in species growth rates, reproduction success, survival rates, and functional diversity (Edwards et al., 2014;Raine et al., 2018;Soto et al., 2019). Microevolutionary events influencing phenotypic traits can be observed in less than 10 generations after the triggering event (Soto et al., 2019). Land conversion, such as logging, forest fires, increased agriculture, excessive use of pesticides and herbicides, the introduction of invasive or genetically modified species, and human settlements, can create innumerable stimulus stressors, which might result in a microevolutionary event.
These microevolutionary events can be observed by changes in traits in those species resilient to environmental alterations. Traits are measurable characteristics that affect an individual's fitness, such as body size or shape. The environment might contribute to a measurable change in traits (Hernández et al., 2011). Traits can be morphological, such as body size and shape or length of body parts can be measured more simply (Soto et al., 2019). Behavioral traits can also be important. Examples include diurnal and nocturnal activity, predatory behavior (Larsen et al., 2009;Hernández et al., 2011;Martín et al., 2021), or dung processing behavior (Milotić et al., 2019).
Phenological traits like the effects of seasons in the abundance or life cycles (Lobo & Cuesta, 2021;Daoudi et al., 2022;Martínez et al., 2022). Chemical traits, for instance, chemical attracts that signal predators (Goolsby et al., 2017;Martín et al., 2021). These measures and comprehension of trait diversity help us to understand and evaluate the ecosystem functioning (processes and services) and can be applied to improve decision-making for conservation and ecosystem restoration (Cadotte et al., 2011;Gagic et al., 2015). Dung beetles are highly sensitive to environmental changes. For example, pastures present dung beetle species adapted to forest habitats with some notable environmental changes, including higher temperatures, decreased humidity, dry compact soil, types of available substance, and higher risk of predation (Barragán et al., 2014;Gómez et al., 2018). Besides, phenotypic changes by microevolution have been evaluated in Onthophagus species related with the emergence of horns and the increased of larval survival depending on association with microbiome, which has allowed them to adapt to different niches and environments over time (Hu et al., 2020). As well as, the changes in color polymorphism in Onthophagus proteus driven by environmental factors as altitude (Stanbrook et al., 2021). However, it is important to note that these are recent discoveries and need more study time (Hu et al., 2020).
Changes in body size traits may not be uniform within species in response to the same environmental changes. Males and females are sexually dimorphic, with females being larger than males (Stillwell et al., 2010). Over time, we can find evidence of the advantages of size in individual fitness, such as mating success, fecundity, or growth. For example, a large body size promotes sexual success and fecundity (Blanckenhorn, 2005). However, small male individuals also have advantages because they require less food in ecosystems with low food availability consequently, they are successful in finding a mate and reproducing (Blanckenhorn, 2005;Stillwell et al., 2010).
Likewise, larger females have high fecundity producing many offspring during their life (Stillwell et al., 2010). Morphological changes include food selection which may vary depending on sex, age, or physiological condition (Salomão et al., 2022). Therefore, plasticity of sexual dimorphism concerning body size can differ between sexes and could depend on environmental variables, creating different patterns in sexual dimorphism in different population subject to different environments (Teder & Tammaru, 2005;Stillwell et al., 2010).
Environment plays a vital role in adjusting a particular insect morphological trait to occupy a niche. In arid environments, to reduce desiccation, flightless species exhibit rounder, truncated elytra in comparison with flying species (Stanbrook et al., 2021). Adults of dung beetles body size decreased with increasing temperature throughout development. This decreases energetic costs and helps beetles conserve energy for feeding, reproduction, and immune responses (Carter & Sheldon, 2020;Mamantov & Sheldon, 2021).
Likewise, variation in the hind leg and eye size is consistent with temperature exposure, resource availability, and habitat structure.
Large-eyed individuals of dung beetles are abundant in logged forests that old growth forests (Raine et al., 2018). Additionally, the coloration of the body of dung beetles responds to altitudinal change. All these modifications are mechanisms for thermoregulation (Stanbrook et al., 2021). The principal traits related to dung beetles ability to remove dung are body mass and body size. The effectiveness of dung burial is influenced most by traits related to the prothorax (volume, pronotum length, and width) along with the protibial area is important (deCastro-Arrazola et al., 2020). These traits consider the key dung exploitation strategies, especially the tibial shape, which is an evolutionary marker that changes in relation to competition or adaptation strategy (Macagno et al., 2016).
Differences have also been seen in females of Dichotomius, where they have been found individuals with four protuberances in disturbed areas and two protuberances in forested areas (Pardo-Diaz et al., 2019). Although, the species are known to be habitat generalists and have preferences for forest edges and grasslands as they prefer cow and horse feces (Amat-Garcia et al., 1997;Amézquita et al., 1999;Sarmiento-Garcés & Amat-García, 2009b).
In this study, we investigated the effects of the forest conversion into grassland through the functional morphology of dung beetles Dichotomius problematicus. Habitat conversion can induce differentiation in the functional morphology of resilient species. Therefore, we have set the following aims: (1) to estimate the effect of the conversion over morphological traits and sexual dimorphism of D. problematicus; (2) to compare the phenotypic differences between individuals inhabiting native forest and grassland. We expect differences between individuals of both habitats according to body size and shape. In addition, we expect to find a positive relationship between the size of individuals and habitat, particularly in forest, because there is a greater availability of food resources.

| Study species: Dichotomius problematicus
The genus Dichotomius (Scarabaeidae: Scarabaeinae) are endemic dung beetles from the New World, consisting of approximately 170 named species (Schoolmeesters, 2021) that are distributed from the northeastern United States to central Argentina and the highest diversity is found in tropical South America (Nunes & Vaz-de-Mello, 2020;Rossini & Vaz-de-Mello, 2020). They are paracoprids and the genus is divided into four subgenera: Dichotomius s. str.; Luederwaldt, 1929;Selenocopris Burmeister, 1849 and Luederwaldtinia Martínez, 1951 Nunes & Vaz-de-Mello, 2020). The length of their body usually measures between 8 and 26 mm. Due to large size and nidification habit, they efficiently remove larger amounts of dung from the soil surface than rollers and dwellers (Monteiro et al., 2020). Their eggs and food are deposited beneath the soil surface in a gallery complex, where deposition depth depends on species, type of soil, and environmental temperature (Hanski & Cambefort, 1991;Macagno et al., 2016;Monteiro et al., 2020). (Rossini & Vaz-de-Mello, 2020)is in the IUCN category of Data Deficient (DD) (Vaz-de-Mello et al., 2014).

Dichotomius problematicus
There is a lack of information basic about the specific ecological or population dynamics of D. problematicus. More research is needed for taxonomic verification of the status of the species or subspecies and to make a more informed assessment based on its distribution, population dynamics, and life history patterns. Within Ecuador Dichotomius, species have been reported in Loja (Piscobamba), Morona Santiago, Napo, Pastaza, Sucumbíos, Tungurahua, and Zamora Chinchipe (Chamorro et al., 2019). Dichotomius problematicus is widely distributed in Colombia, Ecuador, and Peru.

| Study area
The study was carried out in the Podocarpus National Park (PNP), located between the provinces of Zamora Chinchipe and Loja, south of Ecuador. The PNP has an altitudinal range from 900 to 1600 m a.s.l. and comprises an area of 146,280 hectares. The region has a humid climate >90% with mean annual precipitation ranging from around 2000 mm to 4500 mm from 1050 mm at 3060 m a.s.l. (Moser et al., 2007). Precipitation exhibits a bimodal pattern with a significant rainy season from April to July and a less intensive rainy season from September to December (Bendix et al., 2006).
Annual mean air temperature decreases with increasing elevation from 19.4°C to 9.4°C (Moser et al., 2007). Podocarpus National Park is considered a megadiverse area due to its high degree of endemism and the number of species that it harbors (Thormann et al., 2018).
Sampling was conducted at the same time between November 2013 and April 2014, for which two habitats were selected: (1) Forest: located in the Bombuscaro (4°06′50″ S, 78°58′00″ W, 950 m a.s.l.), within the PNP in the province of Zamora Chinchipe, and (2) Grassland: located in Timbara (4°03′48″ S, 78°54′42″ W, 1100 m a.s.l.), within the PNP in the province of Zamora Chinchipe. It has an area of 12,750.61 hectares, of which the majority are areas of extensive livestock production. Another part is used for agricultural or subsistence production. This area has a high degree of anthropic intervention due to agricultural activities and the advance of the agricultural frontier.

| Sampling coprophagous beetles
At each habitat site forest and intensive agriculture, we established 20 sampling points distributed along a transect and separated from each other every 50 meters (Larsen & Forsyth, 2005).
At each sampling point, four pit-fall traps were installed and distanced from each other by 1 m. The pit-fall traps consist of a plastic container placed on the ground level and filled one-third of the way with a mixture of water and detergent. Traps were baited with pig feces (20 g) to attract coprophagous species and were left open for 48 hours. Both habitat sites were sampled the same day. The beetles were preserved in a 90% alcohol solution and subsequently identified at the most specific taxonomic level through taxonomic keys (Chamorro et al., 2018(Chamorro et al., , 2019. The col-

| Morphometric measurement
To estimate the functional traits of the Dichotomius problematicus,  (Soto et al., 2019). In addition, the individuals were sexed, using the shape of the tibial spur and genitalia characters depending on which trait or combination of traits was most useful. An Olympus SZ61 stereoscope with micrometric ocular measurement was used to measure the morphological features in millimeters. The sphericity was determined through the formula suggested by Sneed and Folk (Sneed & Folk, 1958) (Soto et al., 2019): 3 √ SI ∕ L 2 where L total body length (long axis), I is elytra width (intermediate axis), S is body thickness (shaft).

| Data analysis
All the analyses were carried out with R statistical program (R-Core- analysis was calculated with the MASS statistical package (Venables & Ripley, 2002).
Finally, the level of significance was established for each of the morphological features in relation to two explanatory variables: (1) habitat (forest and grassland) and (2) sexual dimorphism (male and female) for which a generalized linear models (GLM's) were used, through the glm function with a Gaussian distribution with the link function "identity." Generalized linear models were calculated with the MASS statistical package (Venables & Ripley, 2002). A chisquared test was performed to evaluate the significance of the explanatory variables: habitat and sexual dimorphism.

| RE SULTS
In total, 269 individuals were measured (47 in forest and 222 in grassland) ( Table S1). The average values of size in relation to habitat and sexual dimorphism are similar. However, the total length (L) presents the highest value for both forest and grassland and between females and males (Table 1; Figure 3a,b). The spherecity is higher in grassland but is not different between males and females (Table 1; Figure 4a,b).
Regarding the type of habitat, the NMDS (stress = 0.2) and the PERMANOVA separated the individuals (F = 16.53, r 2 = .055, p < .001) (Figure 2). Sexual dimorphism was not observed (F = 0.523, r 2 = .002, p = .690) nor is there any apparent interaction between habitat and sex (F = 0.5797, r 2 = .002, p = .583). The ordering of the NMDS shows that the morphological traits measured are partially overlapping with a significant subset of the beetles from grasslands differing from beetles in forest habitats (Figure 2a). However, when comparing traits for D. problematicus with NMDS, traits are completely overlapping between male and female beetles (Figure 2b).
These patterns show that there are greater morphological differences (10 of 13 variables) associated with habitat than with sexual dimorphism (four of 13 variables) ( Table 1).
The results of the PCAof the 13 morphological variables we impute five were responsible for the largest degree of variation in the data (PC1 and PC2). Those five variables were consistent with contribution of variables we selected body thickness (S), pronotum width (PW), pronotum length (PL), head width (HW), and elytra width (I) that explain the 72.3% of variation (Figure 7a,b).
These results show a considerable effect on morphological traits in the conversion from forest into grassland on the individuals of D. problematicus (Tables 1 and 2). The individuals from the forest are longer (Figure 3a) and less spherical than those of grassland (Table 1; Figure 4a). However, the individuals from grassland tend to be smaller ( Figure 3a) but with protibia length and width larger ( Figure 5c,b) as well as head longer ( Figure 5a) and pronotum robust than forest individuals (Figure 6a-c). With respect to dimorphism sexual, the females tend have longer metatibia than males ( Table 1).
In relation with the difference between males and females under the grassland-forest factor conditions, we found greater variation in traits associated with grassland: pronotum length (PL), protibia width (pTW), metatibia length (mTL), and sphericity (Sph) with respect to forest with only two traits: body thickness (S) and sphericity (Sph) ( Table 2). Despite some previous publications suggesting that males particularly paracorprids are larger, we did not find this to be true in our study area (deCastro-Arrazola et al., 2020). Total length (L) represents a proxy for size and indicates that individuals in the forest are larger than individuals in the grasslands.

F I G U R E 2
This could be a result of the quantity and quality of food resources, available in pastures when compared with forests like the pattern observed in C. quinquemaculatus (Soto et al., 2019). Grasslands offer an abundance of cow dung that is exposed to more extreme temperature and humidity changes than in the forest. In contrast, the forests will be relatively poor in dung recourses because there are no mammals remotely as large or abundant as cows and thus offer less dung but a greater diversity of resources, possibly of higher quality. This might result in better quality nutrients for developing beetles and more choice for high-quality nutrients for parents.
Thus, the larger-sized dung beetles are more sensitive to ecological disturbance, giving the opportunity to smaller individuals in open areas (Shahabuddin et al., 2010) or conversely there are fewer resources available to feed offspring, and they are unable to reach the size of beetles in the forest. The use of pit-fall traps does not allow us to characterize any changes in behavior including possible increased competition for resources due to increased D. problematicus individuals in the pasture. We do acknowledge that there is clearly a greater density of D. problematicus in the grassland. However, it should not be discounted without further investigation that beetles of smaller size might result from the increased competition.
As well as, in grassland, we found individuals had longer measured, (1) head width (HW), (2) protibia width (PW), and (3)  Spherecity (Sph) could be associated with reproductive capacity (Raine et al., 2018) and is relatively higher in females in our study.
Sphericity is also associated with tolerance to heat in open areas being less spherical than forest (Soto et al., 2019). Elytra length could be related to flying efficiency, dispersal distance, and to being higher in forest. It would be shorter in males because they might not TA B L E 2 Mean and standard error values of the morphological traits of Dichotomius problematicus concerning sexual dimorphism in function of habitat.

TA B L E 3 (Continued)
experience the same pressure to fly longer distances to provide for their offspring (Raine et al., 2018).
The pronotum is larger in individuals of grassland that forest possibly because this part of the body are inserted legs and wings and investing more in pronotal size could increase dispersal capacity (Nunes et al., 2021). The food in nature is a limit resources (Hanski & Cambefort, 1991;Verdú & Galante, 2002), due to its patchy distribution, but high more importantly quality food resources are more important than just food resources. Exposure to wind, sun, and other competitors can reduce resource quality and availability (Hanski & Cambefort, 1991;Menéndez et al., 2014;Salomão et al., 2022).
Therefore, dung beetles must arrive quickly and, in some cases,

F I G U R E 3
Effect of habitat (forest and grassland) (a) and sexual dimorphism (female and male) (b) the total length of Dichotomius problematicus, in a Tropical Forest in Ecuador (mean ± 95% confidence intervals).

F I G U R E 4
Effect of habitat (forest and grassland) (a) and sexual dimorphism (female and male) (b) on sphericity of Dichotomius problematicus, in a Tropical Forest in Ecuador (mean ± 95% confidence intervals).

F I G U R E 5
Effect of habitat (forest and grassland) on head width (a), protibia width (b) and protibia length (c) of Dichotomius problematicus, in a Tropical Forest in Ecuador (mean ± 95% confidence intervals).

F I G U R E 6
Effect of habitat (forest and grassland) on pronotum high (a), pronotum length (b) and pronotum width (c) of Dichotomius problematicus, in a Tropical Forest in Ecuador (mean ± 95% confidence intervals).
relocate the food sources to ensure their survival and that of their offspring (Hanski & Cambefort, 1991;Salomão et al., 2022).
All these changes in the species assemblage, structure, and morphology drive changes in body size, abdomen size, wing loading, hind leg size, and eye size (Raine et al., 2018), which change a species' ecological functions and their efficacy with regard to ecosystem services (functional traits) such as the quantity of manure removal (Slade et al., 2007), seed dispersal ability (Nichols et al., 2008), and biological control of parasites (Nichols et al., 2008). If a functional group (dwellers, tunnelers, or rollers) is absent greater, it is expected to exacerbate a loss or degradation of ecosystem services, which could affect the rest of the ecological network (Gardner et al., 2008;Slade et al., 2007).
With all this evidence, we suggest that functional diversity based on traits is an important tool that complements the taxonomical diversity because traits reflect the state of an ecosystem at a particular time and individuals' response to the current state.
These responses by the dung beetle community have downstream effects on ecosystem services such as how efficiently dung beetles will be as seed dispersers and the speed and quantity of dung removal (Gagic et al., 2015;Milotić et al., 2019;Slade et al., 2007).
warming because an increase in mean minimum temperature could affect the structure small individuals who will be more sensitive to these environmental changes and migrate or disappear (Milotić et al., 2019). This trait of dung beetles improves their usefulness as indicators of anthropogenic habitat disturbance, on global and local levels (Gagic et al., 2015;Hernández et al., 2011;Milotić et al., 2019).
Over this study, we found patterns of changes in the size of beetles and the structural morphology of their anterior legs, head, and pronotum changed with respect to habitat and, to a lesser degree sexual dimorphism. Thus, individuals of D. problematicus reflect the conservation state of an ecosystem in the south tropical forest of Ecuador where our study was conducted. Future studies should focus on investigating the microevolutionary adaptation of dung beetles addressing different habitats to improve understanding of these processes of adaptation and assess whether these changes respond in a different way to other stressors, and deepen the knowledge of their biology and ecology.

ACK N OWLED G M ENTS
We would like to thank David Donoso and Sebastian Padron for the comments on the manuscript; to Israel Cartuche for assistance with fieldwork. Podocarpus National Park Administration and the Ministerio del Ambiente, Agua y Transición Ecológica from Ecuador for the permissions to collect. Finally, we would like to thank the REASSEMBLY research unit and DFG.

FU N D I N G I N FO R M ATI O N
This research received no external funding.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are available in the supplementary material of this article.

PE R M I SS I O N TO R E PRO D U CE M ATE R I A L S FRO M OTH E R S O U RCE S
None.